RESUMEN
We present a case of a 75-year-old woman who presented with progressive kidney failure. Kidney biopsy performed to determine the cause of kidney failure showed amyloidosis of undetermined type. Laser microdissection of the Congo Red-positive glomeruli followed by mass spectrometry studies showed a large number of spectra matching apolipoprotein E, serum amyloid P component, and gelsolin, consistent with a diagnosis of gelsolin-associated renal amyloidosis. Sequencing of the gelsolin gene revealed a previously undescribed sequence variant, a guanine to adenine substitution at nucleotide 580 of the coding sequence, corresponding to a predicted glycine to arginine mutation at amino acid 194. Gelsolin amyloidosis typically involves the nerves and skin, with only rare reported involvement of the kidney. An atypical finding on electron microscopy was that of a swirling pattern of the amyloid fibrils. The novel gelsolin variant may be responsible for the unusual clinical and pathologic presentation. The report also highlights the usefulness of laser microdissection and mass spectrometry in the typing of difficult cases of amyloidosis.
Asunto(s)
Amiloidosis/metabolismo , Gelsolina/genética , Gelsolina/metabolismo , Variación Genética/genética , Enfermedades Renales/metabolismo , Anciano , Secuencia de Aminoácidos , Amiloidosis/complicaciones , Amiloidosis/patología , Biopsia , Femenino , Gelsolina/análisis , Humanos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/complicaciones , Enfermedades Renales/patología , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación/genética , Insuficiencia Renal/etiologíaRESUMEN
GOALS: To underscore the utility of DNA fingerprinting for clarifying disparate results from endoscopic pathologic specimens. BACKGROUND: Occasionally, serially obtained gastrointestinal biopsies may yield inconsistent results. These discrepancies pose a dilemma for gastroenterologists and their patients, especially when malignancy is a consideration. STUDY: Patients referred to our tertiary care center from outside institutions had undergone endoscopically obtained esophageal biopsies showing malignancy, verified by pathologists at both our site and from the referring center. Repeat endoscopic biopsies at our center did not show malignancy. To verify that different sets of biopsies came from the same patient, we performed a polymerase chain reaction-based analysis comparing the 2 specimens. This analysis, called DNA fingerprinting, can show a high degree of certainty whether 2 specimens came from the same patient. RESULTS: In each case, DNA fingerprinting verified a match, laying the groundwork for intervention. One patient underwent endoscopic radiofrequency ablation to the esophageal mucosa involved. Another underwent esophagectomy with partial gastrectomy. Both are doing well clinically and remain cancer-free on follow-up. CONCLUSIONS: DNA fingerprinting is a powerful and a relatively inexpensive tool. Usually, only small amounts of tissue are required, and even degraded or archival tissue is adequate. DNA fingerprinting can be an important tool in the gastroenterologist's arsenal to help clarify conflicting results, allowing the patient and physician to move forward with the management.
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Dermatoglifia del ADN/métodos , Endoscopía Gastrointestinal/métodos , Neoplasias Gastrointestinales/diagnóstico , Biopsia , Ablación por Catéter/métodos , Esofagectomía/métodos , Estudios de Seguimiento , Gastrectomía/métodos , Neoplasias Gastrointestinales/patología , Neoplasias Gastrointestinales/cirugía , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Derivación y ConsultaRESUMEN
Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.
Asunto(s)
Distrofia Endotelial de Fuchs/genética , Amplificación de Genes , Predisposición Genética a la Enfermedad , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido , Alelos , Biología Computacional/métodos , Distrofia Endotelial de Fuchs/diagnóstico , Edición Génica , Estudios de Asociación Genética , Genómica/métodos , Genotipo , Humanos , Fenotipo , ARN Guía de Kinetoplastida , Repeticiones de TrinucleótidosRESUMEN
Purpose: CTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD. Methods: Peripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects. Results: The STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression. Conclusions: Our findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.
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Distrofia Endotelial de Fuchs/genética , Regulación de la Expresión Génica/fisiología , ARN Mensajero/genética , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido , Adulto , Anciano , Anciano de 80 o más Años , Southern Blotting , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
PURPOSE: To investigate single nucleotide polymorphisms (SNPs) and trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene in a large cohort of German patients with Fuchs endothelial corneal dystrophy (FECD). METHODS: Genomic DNA was obtained from 398 patients with FECD and from 58 non-FECD controls. Thirty-seven previously reported SNPs were evaluated by genotyping. The 398 FECD samples were analyzed for TNR expansions by short tandem repeat assays and Southern blotting. The possible associations between the TNR length and clinical parameters (age, sex, visual acuity, and central corneal thickness) were analyzed in 132 patients. RESULTS: The SNPs in COL8A2, TCF8, LOXHD1, and AGBL1 showed no heterogeneity in 36 cases, although SLCA411 showed 3 nonsense mutations. SNPs were detected for TCF4 (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), but only rs613872 showed a significant association with FECD (P = 9.93 × 10). Overall, 315/398 (79%) patients harbored TNR lengths >50, whereas no non-FECD controls harbored TNR lengths >50. The TCF4 SNP rs613872 genotype was TT: 39 (67%), TG: 18 (31%), and GG: 1 (2%) in non-FECD controls; TT: 39 (47%), TG: 38 (46%), and GG: 6 (7%) in FECD cases harboring TNR <50; and TT: 23 (8%), TG: 224 (79%), and GG: 38 (13%) in FECD cases harboring TNR >50 (P = 2.93 × 10). No significant association was detected between the TNR length and clinical parameters. CONCLUSIONS: Our large German cohort demonstrated a significant association between the risk allele G in rs613872 and FECD, irrespective of TNR expansion, although this risk allele was more frequent in FECD cases with TNR expansion than without.
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Distrofia Endotelial de Fuchs/genética , Predisposición Genética a la Enfermedad , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
Purpose: To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD). Methods: Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system. Results: Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins. Conclusions: Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Distrofia Endotelial de Fuchs/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Empalme del ARN/genética , ARN Mensajero/genética , Factores de Transcripción/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Southern Blotting , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , Femenino , Distrofia Endotelial de Fuchs/diagnóstico , Distrofia Endotelial de Fuchs/metabolismo , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Factor de Transcripción 4 , Factores de Transcripción/metabolismo , Expansión de Repetición de TrinucleótidoRESUMEN
Decline in mitochondrial DNA (mtDNA) copy number, function, and accumulation of mutations and deletions have been proposed to contribute to age-related physical decline, based on cross sectional studies in genetically unrelated individuals. There is wide variability of mtDNA and functional measurements in many population studies and therefore we assessed mitochondrial function and physical function in 18 families of grandmothers, mothers, and daughters who share the same maternally inherited mtDNA sequence. A significant age-related decline in mtDNA copy number, mitochondrial protein expression, citrate synthase activity, cytochrome c oxidase content, and VO2 peak were observed. Also, a lower abundance of SIRT3, accompanied by an increase in acetylated skeletal muscle proteins, was observed in grandmothers. Muscle tissue-based full sequencing of mtDNA showed greater than 5% change in minor allele frequency over a lifetime in two locations, position 189 and 408 in the noncoding D-loop region but no changes were noted in blood cells mtDNA. The decline in oxidative capacity and muscle function with age in three generations of women who share the same mtDNA sequence are associated with a decline in muscle mtDNA copy number and reduced protein deacetylase activity of SIRT3.
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ADN Mitocondrial/fisiología , Mitocondrias Musculares/fisiología , Actividad Motora/fisiología , Fuerza Muscular/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Adolescente , Adulto , Factores de Edad , Anciano , Femenino , Humanos , Persona de Mediana Edad , Fosforilación Oxidativa , Conducta Sedentaria , Análisis de Secuencia de ADN , Sirtuina 3/metabolismo , Adulto JovenRESUMEN
This paper presents an overview of the conclusions from an international conference convened to address current issues related to the provision of Cystic Fibrosis carrier screening within Europe. Consensus was not aimed at stating whether such a programme should be implemented. Instead the focus was to provide a framework for countries and agencies who are considering or planning its establishment. The general principles and target population of Cystic Fibrosis carrier screening, advantages and disadvantages, health economics, monitoring and future evaluative and research directions were covered. A range of screening strategies have been assessed and compared: pre-conceptional and prenatal screening; individual and couple screening; sequential and simultaneous sampling or testing. Furthermore, technical issues were examined with respect to the choice of the panel of mutations, its detection rate, sensitivity, management of intermediate 'at-risk' couples, screening approach to different populations and ethnic minorities, and assurance of laboratory quality control. The consensus statement also aims to establish the benchmarks for communicating with health care providers, the general public and potential and actual participants before and after the genetic test.